Field of the Invention

The present invention relates to cellulose beads, processes for preparing such beads and their various uses. In particular, the present invention relates to 2,3-dialdehyde cellulose (DAC) beads and their preparation via a process comprising the step of oxidizing a cellulose substrate.

Background to the Invention

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or common general knowledge.

Since their preparation was reported for the first time in the early 1950s, cellulose beads have become an important class of industrial materials (see, for example, Gericke et al., Chem Rev, Vol. 1 13 pp. 4812-4836 (2013) and Wolf et al., Pharmazie, Vol. 47 pp. 121- 125 (1992)).

The preparation of cellulose beads is generally a tedious multi-step process. Although different methods for their preparation have been developed, they all generally rely on some type of phase inversion involving the same basic steps, namely:

(1 ) dissolution of the cellulose in a suitable solvent (e.g. organic solvents or ionic liquids);

(2) atomization and/or surfactant aided dispersion; and

(3) coagulation and solidification of the droplets formed in a non-solvent (i.e. a so- called coagulant solution).

Each of the above-mentioned steps above may pose problems. For example, dedicated solvents are required in order to dissolve the cellulose and, in some instances, chemical modifications of the cellulose may be required in order to make it soluble. However, many of the solvents commonly used for dissolving the cellulose are toxic, flammable, chemically aggressive and/or expensive (implying that they would need to be regenerated). Moreover, in the second step, formation of droplets of the dissolved cellulose phase is critical and often requires sophisticated equipment and rigorous control to produce the desired droplet size. Typically, there is a trade-off between the amount of solvent needed to dissolve the cellulose and the viscosity of the resulting solution since, on one hand, it is desirable to limit the volume of the solvent used to dissolve the polymer while, on the other hand, concentrated, highly viscous liquids are hard to atomize.

Although there are a number of methods for droplet formation (including dropping, jet cutting, spinning drop atomization, rotating disc or ultrasonication) atomization of viscous cellulose solution into small (1 to 10 μιτι) droplets is difficult and, therefore, surfactant- aided dispersion techniques are often needed. If further modification of the cellulose is required, it can generally be performed only after the formation and rigorous purification of the beads.

The use of periodate in glycol cleavage reactions, to selectively oxidize the vicinal hydroxyl groups in the C-2 and C-3 position of cellulose to the corresponding aldehydes with the concomitant cleavage of the C-2-C-3 bond, is one of the most potent methods for modification of cellulose (Perlin, Advances in Carbohydrate Chemistry and Biochemistry, Vol 60, pp. 183-250 (2006)). The reaction allows for surface oxidation of cellulose to produce 2,3-dialdehyde cellulose (DAC), which has a number of interesting applications (for example in protein immobilization, chromatography, drug delivery and graft co-polymerization). However, this reaction does not produce cellulose beads; in particular, cellulose beads having high levels of homogenous oxidation.

Previously, the use of periodate as a stoichiometric oxidizing agent has not been sustainable on an industrial scale due to the consumption of large quantities of periodate. However, recent investigations have demonstrated the possibility of regenerating the periodate either electrochemically or by using low-cost chemical oxidants; thus making periodate oxidation a viable option even for large-scale applications.

Nevertheless, a high degree of oxidation of highly crystalline cellulose (e.g. cellulose from Cladophora green algae) has proved to be difficult to achieve due to the high chemical inertness of this cellulose substrate. Although high concentrations of periodate (corresponding to 10.7 equivalents of sodium metaperiodate per anhydroglucose unit of Cladophora cellulose) and long reaction times (of up to 260 hours) have been employed, the degree of cellulose oxidation achieved has not exceeded 30% (Kim et al., Biomacromolecules, Vol. 1 pp. 488-492 (2000)). Importantly, such processes have not allowed for the production of cellulose beads. We have now unexpectedly found that cellulose beads may be prepared using a one- step, one-pot method utilizing oxidizing agents in water; thus eliminating the need to use organic solvents or ionic liquids to dissolve the cellulose, atomization equipment or surface-active dispersion aids for droplet formation, and/or coagulation solutions. Surprisingly, contrary to the data previously reported, it was found that not only is it possible to achieve complete oxidation of the 2,3-hydroxyl groups in highly crystalline cellulose but also that the morphological and structural properties of the latter can be tailored so as to produce fully oxidized DAC spherical beads in a one-pot process. Disclosure of the Invention

In a first aspect of the invention, there is provided a process for the preparation of cellulose beads comprising the step of reacting a cellulose substrate with an oxidant, which process may be referred to herein as the process of the invention.

All individual features (e.g. particular embodiments) mentioned herein may be taken in isolation or in combination with any one or more other feature (e.g. one or more other particular embodiments) mentioned herein. The skilled person will understand that the reaction of the cellulose substrate with an oxidant may be performed in a solvent. In particular, the skilled person will understand that the reaction may be performed in a polar solvent, which solvents are well known to those skilled in the art. Particular polar solvents that may be employed in the reaction of the cellulose substrate with an oxidant include dimethyl sulfoxide (DMSO), acetone, ethyl acetate, acetonitrile, dimethyl formamide (DMF), ionic liquids, methanol, ethanol and water.

In a particular embodiment, there is provided a process for the preparation of cellulose beads comprising the step of reacting a cellulose substrate with an oxidant in water. The skilled person will understand that the reference to reaction of the cellulose substrate with an oxidant in water may refer to oxidation of the cellulose substrate in the form of dispersion in water (i.e. an aqueous dispersion). Thus, the process of the invention may be referred to as a process for the preparation of cellulose beads comprising the step of reacting a cellulose substrate with an oxidant, wherein the cellulose substrate is present as a dispersion in water.

Alternatively, the process of the invention may be referred to as a process for the preparation of cellulose beads comprising the step of reacting an aqueous dispersion of cellulose with an oxidant.

In a particular embodiment, there is provided a process for the preparation of cellulose beads comprising the steps of:

(i) forming a dispersion of cellulose in water (which may be referred to as an aqueous dispersion of cellulose);

(ii) reacting the cellulose with an oxidant, optionally in the presence of a pH buffering agent; and

(iii) recovering the cellulose beads.

In a more particular embodiment, there is provided a process for the preparation of cellulose beads comprising the steps of:

(i) forming a dispersion of cellulose in water;

(iia) reacting the cellulose with an oxidant, optionally in the presence of a pH buffering agent;

(iib) quenching the oxidant; and

(iii) recovering the cellulose beads.

The skilled person will understand that steps (i) to (iii) as defined in the above-mentioned embodiments are intended to be performed in the order indicated.

In more particular embodiments, the process of the invention may consist essentially of steps (i) to (iii) as defined in the above-mentioned embodiments. As discussed above, the process of the invention requires reaction of a cellulose substrate with an oxidant in water, which the skilled person will understand as indicating that water is used as a solvent (i.e. the reaction medium) for the reaction (i.e. the oxidation).

In a particular embodiment, the process of the invention may be performed using water (e.g. deionized water) as the sole (i.e. only) solvent.

In an alternative embodiment, the process of the invention may be performed in water comprising one or more additional solvent (i.e. one or more solvent other than water). In particular, the process of the invention may be performed in a solvent system (i.e. a mixture of solvents) comprising water and one or more additional solvent.

For the avoidance of doubt, references herein to the solvent system as "water" may refer to a solvent consisting of only water, or to a solvent system comprising water as described herein, as the case may be.

Such additional solvents may include solvents that are capable of acting as radical scavengers (i.e. solvents capable of reacting with and thus deactivating radical species, such as oxygen radicals). Particular additional solvents (i.e. radical scavengers) that may be mentioned include alcohols, including but not limited to propan-1-ol.

The skilled person will understand that the step of reacting the cellulose substrate with an oxidant as described herein may also be referred to as oxidizing the cellulose substrate. As used herein, the reference to an oxidant (i.e. an oxidizing agent) will be understood to refer to an agent capable of oxidizing a vicinal diol moiety (i.e. a 1 ,2-dihydroxy moiety) to form corresponding aldehyde moieties (i.e. through oxidation of the hydroxyl groups and cleavage of the associated carbon-carbon bond). Particular oxidants that may be mentioned include:

periodic acid (iodic (VII) acid) and salts thereof; and

lead tetra-acetate (i.e. lead (IV) acetate).

In particular, the skilled person will understand that periodic acid and salts thereof may be used as oxidizing agents in the transformation of vicinal diols to dialdehydes. Moreover, the skilled person will understand that the actual oxidant is not typically the corresponding periodate (ICV). but rather para- or meta-periodate (H„l06 ^(5"n)" ). As referred to herein, the general term periodate may refer to all such oxoiodate species of heptavalent iodine, either neutral or deprotonated. In a particular embodiment, the oxidant (i.e. the agent used for oxidizing the cellulose substrate) is selected from the group consisting of:

periodic acid and salts thereof (such as sodium metaperiodate); and

lead tetra-acetate. In a more particular embodiment, the oxidant (i.e. the agent used for oxidizing the cellulose substrate) is sodium metaperiodate.

The skilled person will understand that in order to achieve high levels of oxidation of the cellulose substrate the oxidant may be employed in a greater than stoichiometric amount in relation to the cellulose substrate. Thus, more than one equivalent of the oxidant may be employed relative to the amount of cellulose substrate.

In a more particular embodiment, the cellulose substrate is reacted with greater than one equivalent of oxidant (i.e. greater than one equivalent of oxidant relative to the amount of cellulose substrate). For example, the cellulose substrate may be reacted with up to about 5 equivalents of oxidant (such as about 2 to about 5 equivalents).

The process of the invention may also comprise regeneration of the oxidant by reaction with an additional oxidant (i.e. a different oxidant to that used for the oxidation of the cellulose substrate), which additional oxidant may be capable of oxidizing the reacted (i.e. reduced) derivative of the oxidant used for the oxidation of the cellulose substrate, which may have the effect of regenerating the oxidant used for the oxidation of the cellulose substrate. In a particular embodiment, following the oxidation of the cellulose substrate, the oxidant used in the oxidation of the cellulose substrate may be reacted with an additional oxidant (i.e. a further oxidant other than the oxidant used in the oxidation of the cellulose substrate), which additional oxidant may serve to regenerate (i.e. restore the oxidizing properties of) the oxidant used in the oxidation of the cellulose substrate. Thus, in relation to embodiments comprising steps (i) to (iii) as described above, the process of the invention may comprise the further steps of:

regeneration of the oxidant by reaction (i.e. of the reacted oxidant, which may be present in solution) with an additional oxidant; and

- recovery of the regenerated oxidant.

Particular additional oxidants that may be mentioned include those capable of oxidizing the reacted (i.e. reduced) derivative of the oxidant used for the oxidation of the cellulose substrate (i.e. those capable of regenerating the oxidant).

For example, where the oxidant used in the process of the invention is a salt of periodic acid (such as sodium metaperiodate), the oxidant may be regenerated by reaction with Oxone ® (potassium peroxymonosulfate) under conditions known to those skilled in the art (for example, as described in US patent number US 6,620,928, the contents of which are incorporated herein by reference).

The level of crystallinity of the cellulose in the cellulose beads obtained using the process of the invention (or any one more embodiment thereof) may be controlled by adjusting the pH of the reaction (i.e. the pH of the solution used in the oxidation of the cellulose substrate). Typically, more acidic conditions will reduce the level of crystallinity obtained in the cellulose beads.

In particular, the pH of the reaction may be varied from slightly acidic (e.g. a pH of about 6) to strongly acidic (e.g. a pH of about 1), with less acidic conditions having a milder effect on the depression of the degree of cellulose crystallinity.

For example, the reaction may be performed at a pH of less than about 6, such as less than about 4 (e.g. less than about 2, such as at a pH of from about 0 to about 2). In a more particular embodiment, the step of oxidizing the cellulose substrate (i.e. reacting the cellulose substrate with an oxidant) is performed at a pH of about 4 to about 6 (such as a pH of about 4.5 to about 5.5, e.g. at a pH of about 4.5 or about 5.5).

The skilled person will understand that the pH of the dispersion may be adjusted and/or maintained through the addition of a buffering agent, which may be added in the form of a buffering solution (e.g. an aqueous solution of a buffering agent). Thus, in particular embodiments (such as those where the pH of the oxidation reaction is varied), the process is optionally performed in the presence of a buffering agent (which may be referred to as a buffering solution or simply as a buffer).

In a more particular embodiment, the solution of the cellulose substrate is buffered at a pH of about 4 to about 6 (such as a pH of about 4.5 to about 5.5, e.g. at a pH of about 4.5 or about 5.5). Such buffering agents (and corresponding buffering solutions) will be known to those skilled in the art and may be selected based on the pH required; for example, a solution buffered at a pH of around 4.5 or around 5.5 may be obtained through addition of a suitable amount of an acetate buffer. As discussed herein, the process of the invention requires reacting a cellulose substrate with an oxidant in water. The skilled person will understand that references to a cellulose substrate herein are intended to refer to the cellulose being reacted (with an oxidant).

The process of the invention is particularly effective in achieving high levels of oxidation when using a cellulose substrate having a high degree of crystallinity. The process of the invention is also particularly effective when performed using native cellulose.

In a particular embodiment, the cellulose substrate is cellulose (e.g. native cellulose) having a high degree of crystallinity (i.e. highly crystalline cellulose, such as highly crystalline native cellulose).

As used herein, the reference to highly crystalline cellulose may be refer to cellulose (e.g. native cellulose) having a degree of crystallinity of at least 60%, such as at least 70% (e.g. at least 80%), which may be measured with X-ray diffraction (XRD).

One skilled in the art will understand that different methods of characterizing the degree of cellulose crystallinity, e.g. FTIR or NMR, may produce a different numerical value. Therefore, the values reported here are method-specific and should not be considered limiting if other methods of characterization are used. In a more particular embodiment, the cellulose substrate is native cellulose having a degree of crystallinity of at least 90%.

Examples of highly crystalline (native) cellulose that may be used as the cellulose substrate include:

algae cellulose, such as cellulose from macroscopic green algae (e.g. those from Cladophorales, including but not limited to Cladophora, Chaetomorpha, Rhizoclonium, Mycrodyction, Siphonocladales, including but not limited to Valonia, Dictyospheria, and Siphonocladus orders), microscopic/planktonic algae (e.g. those from Glaucocystales, including but not limited to Glaucocystis, or Chlorelalles, including but not limited to Oocystis order);

bacterial cellulose, including but not limited to cellulose from Acetobacter, Agrobacterium, and Sarcina; and

native cellulose derived from aquatic animals (e.g. tunnicates, such as Halocynthia).

In a particular embodiment, the cellulose substrate is Cladophora cellulose.

The skilled person will understand that the cellulose substrate may consist of a mixture of one or more type of cellulose (such as one or more highly crystalline cellulose). In a particular embodiment, the cellulose substrate comprises a single type of cellulose (e.g. a single type of highly crystalline cellulose, such as Cladophora cellulose).

The process of the invention (in particular, the process defined in embodiments relating to steps (i) to (iii)) may be performed in a single reaction vessel, which may be referred to as a one pot process.

Thus, in a particular embodiment, the process of the invention is performed as a one pot process. As described herein, the process of the invention comprises the step of reacting a cellulose substrate with an oxidant, which may be referred to as the oxidation reaction.

The oxidation reaction may be controlled (i.e. to achieve the required level of oxidation) by adjusting the conditions employed (such as the temperature and/or duration of the reaction). Such variation of conditions may be employed in order to achieve levels of oxidation of up to 100%. In a particular embodiment, the step of reacting the cellulose substrate with an oxidant (e.g. step (ii) or (iia) as defined in embodiments referred to above) may be performed at room temperature.

As used herein, references to performing a process (i.e. the relevant process step) at room temperature will refer to performing the process without heating or cooling processes, thus resulting in the reaction being performed at ambient temperature (typically about 18 to about 22 °C, such as at about 20 °C).

The period of time for which the reaction between the cellulose substrate and the oxidant is maintained may be varied, for example, in order to achieve the required level of oxidation. In a particular embodiment, the step of reacting the cellulose substrate with an oxidant (e.g. step (ii) or (iia) as defined in embodiments referred to above) may be performed for a period of at least 24 hours (for example, for a period of from about 24 hours to about 500 hours). In a more particular embodiment, the step of reacting the cellulose substrate with an oxidant (e.g. step (ii) or (iia) as defined in embodiments referred to above) may be performed for a period of at least 240 hours (for example, for a period of from about 240 hours to about 500 hours, e.g. for about 240 hours). In a particular embodiment, the step of reacting the cellulose substrate with an oxidant (e.g. step (ii) or (iia) as defined in embodiments referred to above) may be performed in the absence of light (i.e. in the dark).

The reaction of the cellulose substrate may be terminated using techniques known to those skilled in the art, such as by quenching of the oxidant (i.e. as described in step (iib) of the embodiment referred to above).

As used herein, references to quenching of the oxidant will be understood to refer to reaction of any unreacted oxidant (e.g. oxidant that remains unreacted following the step of reacting the cellulose substrate with an oxidant) such that the oxidant is no longer reactive (i.e. is no longer capable of oxidation). In a particular embodiment, the process of the invention comprises the step of quenching the oxidant. For example, the process of the invention may comprise the step of adding a quenching agent to the solution (i.e. following the step of reacting the cellulose substrate with an oxidant).

In a more particular embodiment, the process of the invention comprises the step of terminating the reaction between the cellulose substrate and the oxidant by quenching of the oxidant.

Examples of agents capable of quenching the corresponding oxidant (i.e. quenching agents) will be known to those skilled in the art. Particular quenching agents that may be mentioned include compounds having vicinal alcohols, vicinal amines, or a combination thereof (e.g. 1-hydroxy-2-amino compounds, and the like). More particular quenching agents that may be mentioned include ethylene glycol.

In a particular embodiment (e.g. in relation to step (iib) in the embodiment described above), the process of the invention may comprise the step of quenching the oxidant through the addition of ethylene glycol.

As described herein, the process of the invention results in the production of cellulose beads. Therefore, the process of the invention may comprise (i.e. as a final step) the recovery of those beads. In a particular embodiment, the process of the invention may comprise the step of recovering the cellulose beads (i.e. the cellulose beads formed by the process of the invention).

In a more particular embodiment, the process of the invention may comprise the step of removing the cellulose beads from the dispersion, and optionally washing the recovered beads (e.g. with water).

For example, in the relevant embodiments referred to above, step (iii) may comprise (or consist of) removing the cellulose beads from the solution, and optionally washing the recovered beads (e.g. with water). As described herein, the process of the invention is a process for preparing cellulose beads, which process comprises oxidation of a cellulose substrate.

In particular, the process of the invention comprises oxidation of vicinal diol moieties (i.e. a 1 ,2-dihydroxy moieties) in the cellulose substrate to form corresponding aldehyde moieties (i.e. through oxidation of the hydroxyl groups and cleavage of the associated carbon-carbon bond), which reaction may be referred to herein as oxidation of the cellulose substrate. In particular, the vicinal alcohols oxidized in the process of the invention may be those in the C-2 and C-3 positions of the cellulose substrate.

The skilled person will understand that the process of the invention may result in the formation of cellulose beads comprising 2,3-dialdehyde cellulose. Thus, the process of the invention may be referred to as a process for preparing 2,3-dialdehyde cellulose (DAC) beads.

In particular, the process of the invention may be referred to as a process for preparing 2,3-dialdehyde cellulose (DAC) beads via oxidation of secondary alcohols in a cellulose substrate. More particularly, the process of the invention may be referred to as a process for preparing 2,3-dialdehyde cellulose (DAC) beads via oxidation of secondary alcohols in the C-2 and C-3 positions in a cellulose substrate with concomitant cleavage of the C-2 to C-3 bond. The cellulose (i.e. DAC) beads obtained using the process of the invention may have a degree of oxidation of at least 60% (i.e. 60-100%).

In a particular embodiment, the cellulose (i.e. DAC) beads obtained using the process of the invention may have a degree of oxidation of at least 80% (i.e. 80-100%).

As described herein, the degree of oxidation in the resulting cellulose beads may be controlled by varying the conditions employed in the reaction of the cellulose substrate with the oxidant. The skilled person will understand that the degree of oxidation may be monitored during the reaction using techniques known to those skilled in the art, such as by taking samples (i.e. aliquots) during the reaction and analysis of the aldehyde content of the cellulose contained therein (e.g. by formation of a corresponding Schiff base and elemental analysis thereof).

The process of the invention may produce cellulose (i.e. DAC) beads that are homogenously oxidized. For example, the cellulose (i.e. DAC) beads obtained using the process of the invention may possess at least 80% (e.g. 80 to 100%) homogenous oxidation.

As used herein, references to the cellulose beads being homogenously oxidized will be understood to refer to those oxidation of the cellulose (i.e. the formation of dialdehyde moieties) being evenly distributed throughout each resulting cellulose bead (or a majority thereof).

Thus, the process of the invention may be referred to as a process for preparing homogenously oxidised 2,3-dialdehyde cellulose (DAC) beads via oxidation of secondary alcohols in the C-2 and C-3 positions in a cellulose substrate with concomitant cleavage of the C-2 to C-3 bond.

In particular, the process of the invention may produce cellulose (i.e. DAC) beads that have a homogenously oxidized core, optionally with additional oxidation at the surface (i.e. the surface of the bead).

The process of the invention may produce cellulose (i.e. DAC) beads that are substantially spherical.

In a particular embodiment, the cellulose (i.e. DAC) beads obtained using the process of the invention may be spherical and/or have an average particle size ranging between 0.01 and 100 micron. The process of the invention (in particular, the oxidation reaction, e.g. as described in step (ii) or step (iia) in the embodiments defined above) may comprise stirring of the reaction (for example, stirring using a paddle or a magnetic stirring device).

The person skilled in the art will understand that the particle size of the cellulose beads obtained using the process of the invention can be controlled by adjusting the intensity of stirring during (or immediately after) the reaction of the cellulose substrate with an oxidant.

The process of the invention may allow for the preparation of cellulose beads having varying degrees of porosity.

For example, the process of the invention may be referred to as a process for preparing non-porous 2,3-dialdehyde cellulose (DAC) beads. In particular, the process of the invention may be referred to as a process for preparing non-porous, homogenously oxidised 2,3-dialdehyde cellulose (DAC) beads via oxidation of secondary alcohols in the C-2 and C-3 positions in a cellulose substrate with concomitant cleavage of the C-2 to C- 3 bond.

The person skilled in the art will appreciate that the definition of a porous bead will depend on the accessibility of a given medium to the core of the said material.

As used herein, references to non-porous beads will refer to beads whose core is not accessible to gas molecules, such as inert gas molecules, e.g. air, nitrogen, krypton, argon, or helium, although still accessible to water or other polar molecules. Thus, references to porous beads will refer to beads that allow such permeation.

Non-porous beads will show no or little difference in the bulk porosity, i.e. mass-to- volume ratio, derived from geometrical dimensions and that by using gas permeametry, i.e. so called true density. As a rule of thumb, such non-porous cellulose beads will also show a specific surface area≤ 1 m ² g ^_1 measured using nitrogen gas according to the standard Brunauer-Emmett-Teller (BET) method.

Accordingly, porous beads will show bulk density values, which are substantially smaller than the true density values derived from gas permeametry. As a rule of thumb, porous cellulose beads will typically feature a specific surface area > 1 m ² g ^_1 measured using nitrogen gas according to the standard Brunauer-Emmett-Teller (BET) method.

In a second aspect of invention, there is provided cellulose beads obtainable from (or obtained from) the process of the invention (i.e. the process as described in the first aspect of the invention, or any one or more embodiments thereof). Thus, in one embodiment there is provided 2,3-dialdehyde cellulose (DAC) beads obtainable from (or obtained from) the process of the invention (or any one or more embodiments thereof). In particular, there is provided 2,3-dialdehyde cellulose (DAC) beads obtainable from (or obtained from) the process of the invention (or any one or more embodiments thereof), wherein the cellulose beads have a degree of oxidation of at least 80% (i.e. 80 to 100%).

In a third aspect of the invention, there is provided 2,3-dialdehyde cellulose (DAC) beads having a degree of oxidation of at least 60% (i.e. 60-100%).

In a particular embodiment of the third aspect of the invention, the 2,3-dialdehyde cellulose (DAC) beads may have a degree of oxidation of at least 80% (i.e. 80-100%). In a more particular embodiment of the third aspect of the invention, the 2,3-dialdehyde cellulose (DAC) beads may have a degree of homogenous oxidation of at least 80% (i.e. 80-100%).

In a particular embodiment of the third aspect of the invention, the 2,3-dialdehyde cellulose (DAC) beads may be substantially spherical.

In a more particular embodiment of the third aspect of the invention, the 2,3-dialdehyde cellulose (DAC) beads may be substantially spherical and/or have an average particle size ranging between 0.01 and 100 micron.

For example, the 2,3-dialdehyde cellulose (DAC) beads may have a degree of homogenous oxidation of at least 80% (i.e. 80-100%) and be substantially spherical having an average particle size ranging between 0.01 and 100 micron The cellulose beads obtained using the process of the invention may derivatized, for example, in order to produce porous cellulose beads.

In particular, in order to produce porous cellulose beads, the available aldehyde groups produced by extensive periodate oxidation of the cellulose substrate are reacted with aldehyde protective groups (e.g. by Schiff base reactions with primary diamine-group containing substances). In a fourth aspect of the invention, there is provided as process for the preparation of cellulose (e.g. DAC) beads comprising the steps of:

(a) obtaining cellulose beads using a process as defined in the first aspect of the invention (including any one or more embodiments thereof); and

(b) derivatizing the cellulose beads.

Thus, in a particular embodiment, there is provided a process for the preparation of cellulose beads comprising the steps of:

(i) forming a dispersion of cellulose in water;

(iia) reacting the cellulose with an oxidant, optionally in the presence of a pH buffering agent;

(iib) quenching the oxidant;

(iii) recovering the cellulose beads; and

(iv) derivatizing the cellulose beads.

In a particular embodiment of the fourth aspect of the invention, the cellulose beads produced may be described as being porous cellulose beads. In a particular embodiment, the beads used in step (iv) of the process described above are not dried prior to use in step (iv). Thus, step (iv) may be referred to as derivatizing never-dried cellulose beads.

In a certain embodiment, the process of the fourth aspect of the invention (in particular, comprising steps (a) and (b) as defined above) may be performed as a one pot process.

In a fifth aspect of the invention, there is provided a process for preparing cellulose (e.g. DAC) beads comprising the step of reacting a cellulose bead as defined in the second or third aspect of the invention (including any one or more embodiments thereof) with an agent capable of derivatizing said beads.

In a particular embodiment of the fifth aspect of the invention, the cellulose beads produced may be described as being porous cellulose beads. In a particular embodiment of the fourth and fifth aspects of the invention, the step of derivatizing the cellulose beads (or reacting the beads with an agent capable of derivatizing said beads) may comprise reacting the cellulose beads with one or more aldehyde protecting agent.

Particular aldehyde protecting agents that may be mentioned include agents capable of reacting with aldehyde moieties to form a Schiff base (e.g. an imine or a hydroxyimine), such as an agent selected from the group consisting of 1 ,2-diaminoethane, 1 ,3- diaminopropane, 1 ,4-diaminobutane, 1 ,5-diaminopentane, 1 ,6-diaminohexane, 1 ,7- diaminoheptane, aniline, 1 ,10-phenanthrolin-5-amine, 3-(amino methyl)pyridine, tryptophan and hydroxylamine.

In a particular embodiment of the fourth and fifth aspects of the invention, the step of derivatizing the cellulose beads (or reacting the beads with an agent capable of derivatizing said beads) may comprise reacting the cellulose beads with one or more amine in a reductive amination reaction. Said reductive amination reactions can be performed using techniques that are well known to those skilled in the art; for example, by reaction of the aldehyde with a suitable (e.g. primary) amine (i.e. to form an imine moiety) followed by reduction using a suitable reducing agent (e.g. sodium borohydride).

Particular amines that may be used in reductive amination reactions include 1 ,2- diaminoethane, 1 ,3-diaminopropane, 1 ,4-diaminobutane, 1 ,5-diaminopentane, 1 ,6- diaminohexane, 1 ,7-diaminoheptane, bis-(4-diaminophenyl) ether aniline, o- phenylenediamine, m-phenylenediamine, p-phenylenediamine, 1 ,10-phenanthrolin-5- amine, 3-(amino methyl)pyridine, tryptophan, cysteine and hydroxylamine. In particular, the reaction of cellulose (e.g. DAC) beads with hydroxylamines (e.g. to form a Schiff base) leads to the formation of cellulose beads with a highly smooth surface. The skilled person will understand that the smoothness of the surface of the bead may be determined using techniques known in the art, such as using atomic force microscopy (AFM).

In a particular embodiment of the fourth and fifth aspects of the invention, the step of derivatizing the cellulose beads (or reacting the beads with an agent capable of derivatizing said beads) may comprise reacting the cellulose beads with other agents capable of reacting with the aldehyde moieties in said beads (i.e. DAC beads), such as nucleophiles selected from the group consisting of oxygen nucleophiles (e.g. for hemiacetal and/or acetal formation), nitrogen nucleophiles (e.g. for oxime or hydrazine formation), carbon nucleophiles (e.g. for cyanohydrin formation), bisulfite nucleophiles (e.g. for sulfonate formation) and thiol nucleophiles (e.g. for thioester formation).

Such reactions of the aldehyde groups with nucleophiles may be used to attach functional groups to the cellulose beads (i.e. functional groups bound to the reacting nucleophile).

In this context, the type of the functional group attached to the said DAC beads will have a direct effect on the utility of the beads. Therefore, moieties having a direct biological and/or chemical function (such as those of amino acids, peptides, glycoproteins, lipoproteins, nucleosides, DNA, RNA, porphirines and/or metal chelating ligands) are particularly useful.

In a particular embodiment of the fourth and fifth aspects of the invention, the step of derivatizing the cellulose beads (or reacting the beads with an agent capable of derivatizing said beads) may comprise oxidizing the aldehyde groups to the corresponding carboxyl or carboxylate groups. The skilled person will understand that oxidation of such aldehyde groups (e.g. to the corresponding carboxyl or carboxylate groups) can be performed using techniques (i.e. reagents and conditions) well known to those skilled in the art.

Porous cellulose beads may also be prepared by a process involving exchanging the solvent used in the process of the invention for one or more other solvent, which step may be performed more than once.

Thus, in an alternative fourth aspect of the invention, there is provided a process for the preparation of porous cellulose (e.g. DAC) beads as described in respect of the fourth aspect of the invention (or any one or more embodiment thereof) but wherein step (b) (or, in particular embodiments, step (iv)) is replaced with the step of:

- exchanging the solvent (e.g. water) for another (i.e. different) solvent, and optionally repeating said exchange at least once.

In a particular embodiment, the step of polar-to-non-polar solvent exchange may consist of:

- exchanging the water for a water-miscible (polar) solvent, such as ethanol, methanol, acetone, acetonitrile or ethylacetate; and then exchanging the water-miscible solvent for a volatile water-immiscible solvent, such as diethyl ether, pentane, hexane, benzene, or chloroform; and then evaporating the volatile water-immiscible solvent. One skilled in the art will understand that a variety of a polar-to-non-polar solvent exchanges may also include a so-called critical point drying using liquid carbon dioxide.

The skilled person will understand that the solvent exchange step may be followed by the step of recovering the porous cellulose beads, as described herein. In particular, the step of recovering the porous cellulose beads may be followed by drying of the beads.

In particular, the (or each) step of exchanging the solvent may be performed without prior drying of the cellulose beads. Thus, the step of exchanging the solvent (and the optionally repeated steps) may be performed on never-dried cellulose (e.g. DAC) beads.

In a sixth aspect of invention, there is provided cellulose beads obtainable from (or obtained from) a process as described in the fourth or fifth aspects of the invention (or any one or more embodiments thereof). As described herein, cellulose (e.g. DAC) beads obtainable (or obtained) using a process as defined in the first aspect of the invention (including any one or more embodiments thereof), or as defined in the second or third aspect of the invention (including any one or more embodiments thereof) have numerous uses, for example, in chemical processes and therapeutic applications (i.e. in medicine, such as in biomedical, pharmaceutical, and extracorporeal blood treatment applications).

In a seventh aspect of the invention, there is provided the use of cellulose (e.g. DAC) beads obtainable (or obtained) using a process as defined in the first, fourth or fifth aspects of the invention (including any one or more embodiments thereof), or as defined in the second, third or sixth aspects of the invention (including any one or more embodiments thereof) in:

extracorporeal blood treatment;

immunoadsorption and/or clinical apheresis;

column packing material for liquid chromatography;

affinity chromatography columns for peptide or protein purification;

metal ion-exchange and water purification columns; solid support for catalysts;

carriers for enzyme immobilization;

virus binding/removal agents;

bactericides;

drug delivery vehicles; and/or

sensors.

In particular, there is provided the use of cellulose (e.g. DAC) beads obtainable (or obtained) using a process as defined in the first aspect of the invention (including any one or more embodiments thereof), or as defined in the second or third aspect of the invention (including any one or more embodiments thereof),

wherein the beads have an average particle size of less than 1 micron (e.g. ranging between 0.01 and 1 micron,

in pharmaceutics, diagnostics, and biomedicine as nanocarriers.

Without wishing to be bound by theory, it is thought that the extensive periodate oxidation of highly crystalline cellulose achieved in the process of the invention results in substantial morphological and structural modifications of cellulose. With the increasing level of oxidation, decreasing true density of cellulose and decreasing degree of cellulose crystallinity, the initially fibrous cellulose gradually becomes more compact, as manifested by decreasing N ₂ gas adsorption specific surface area values, and eventually takes on characteristic spherical shapes.

It can be speculated that the bead forming ability of these types of cellulose is related both to reduced stiffness of the elementary fibrils and increased surface hydrophobicity due to the augmented aldehyde content. The transition to the characteristic spherical bead shape is achieved at a degree of cellulose oxidation above 60%, and more preferably above 75% (e.g. above 80%), while the corresponding degree of crystallinity can greatly vary dependi. g on the employed pH.

The present invention may have the advantage that it allows for a one-step method of dialdehyde cellulose (DAC) beads preparation via oxidation of the vicinal alcohols in water without the use of organic dissolving solvents; atomizing equipment or surface- active additives for droplet formation; or coagulation solutions for solidification. The disclosed one-pot procedure for DAC bead preparation in water has several benefits compared to the traditional methods of cellulose bead preparation as it does not require organic solvents or ionic liquids to dissolve cellulose; avoids the use of regenerating coagulant solutions and surfactants or other surface active dispersion aids; and does not employ spraying, atomization or any other droplet forming equipment, thereby significantly facilitating large-scale production. Moreover, the produced beads have a degree of oxidation of 60-100% suggesting a multitude of possibilities for further modifications by utilizing the attractive aldehyde groups to provide the desired functionalized beads with high functional group density.

Summary of the figures

Figure 1 shows SEM micrographs of DAC beads prepared from Cladophora cellulose according to Example 3 (A) DAC beads prepared from bacterial cellulose according to Example 9 (B), Schiff base coupled with hydroxyl amine according to Example 10 (C) and Schiff base coupled with 1 ,7-diaminoheptane according to Example 13 (D).

Figure 2 shows a solid-state ¹³ C NMR spectrum of periodate oxidized Cladophora DAC (240 h) which is in agreement with previously reported DAC NMR spectra.

Examples

The present invention may be illustrated by the following examples. Example 1 - Preparation of DAC beads under unbuffered conditions

Cladophora cellulose, 12 g in 900 mL of deionized water was mixed with 79 g sodium metaperiodate (about 5 mol per mol of anhydroglucose units) dissolved in 900 mL deionized water. The periodate-containing reaction mixture was carefully wrapped in aluminum foil to avoid light and 100 mL of 1-propanol was added to the reaction mixture to serve as a radical scavenger. (Painter, Carbohydrate Research, Vol. 179 pp. 259-268 (1988)) The reaction mixture was vigorously stirred at room temperature in the dark for 10 days. Aliquots were withdrawn after 24, 48, 72, 96, 168 and 240 h (300 mL each time). The withdrawn aliquots were immediately quenched via the addition of ethylene glycol and washed repeatedly with water to provide pure DAC. A total of 6 different samples were produced (for 24, 48, 72, 96, 168 and 240 h, respectively), where the samples subjected to the longest oxidation times (168 and 240 h) produced bead-shaped DAC material.

Example 2 - Preparation of DAC beads at pH 4.5

Cladophora cellulose, 12 g in 900 mL of acetate buffer pH 4.5 was mixed with 79 g sodium metaperiodate (about 5 mol per mol of anhydroglucose units) dissolved in 900 mL acetate buffer pH 4.5. The periodate-containing reaction mixture was carefully wrapped in aluminum foil to avoid light and 100 mL of 1-propanol was added to the reaction mixture to serve as a radical scavenger. The reaction mixture was vigorously stirred at room temperature in the dark for 10 days. Aliquots were withdrawn after 24, 48, 72, 96, 168 and 240 h (300 mL each time). The withdrawn aliquots were immediately quenched via the addition of ethylene glycol and washed repeatedly with water to provide pure DAC. A total of 6 different samples were produced (for 24, 48, 72, 96, 168 and 240 h, respectively), where the samples subjected to the longest oxidation times (168 and 240 h) produced bead-shaped DAC material.

Example 3 - Preparation of DAC beads at pH 5.5 Cladophora cellulose, 12 g in 900 mL of acetate buffer pH 5.5 was mixed with 79 g sodium metaperiodate (about 5 mol per mol of anhydroglucose units) dissolved in 900 mL acetate buffer pH 5.5. The periodate-containing reaction mixture was carefully wrapped in aluminum foil to avoid light and 100 mL of 1-propanol was added to the reaction mixture to serve as a radical scavenger. The reaction mixture was vigorously stirred at room temperature in the dark for 10 days. Aliquots were withdrawn after 24, 48, 72, 96, 168 and 240 h (300 mL each time). The withdrawn aliquots were immediately quenched via the addition of ethylene glycol and washed repeatedly with water to provide pure DAC. A total of 6 different samples were produced (for 24, 48, 72, 96, 168 and 240 h, respectively), where the samples subjected to the longest oxidation times (168 and 240 h) produced bead-shaped DAC material, Figure 1 A shows a SEM micrograph of the sample collected after 240 h and Figure 2 shows the solid-state ¹³ C NMR spectrum of the 240 h sample. Example 4 - Preparation of DAC beads

Cladophora cellulose, 4 g in 200 mL of acetate buffer (pH 5.5) was mixed with 26.4 g sodium metaperiodate dissolved in 400 mL acetate buffer (pH 5.5). The periodate containing solution was carefully kept wrapped in aluminum foil to avoid light. The mixture was stirred at 20 °C in the dark for 10 days. The reaction mixture was divided in 4 aliquotes (150 mL each). For aliquote 1 : any excess periodate was quenched by addition of 20 mL glycerine (added under stirring and stirred for 3 h at rt). After the excess periodate was decomposed, the product was washed by centrifugation using H2O x 6. For aliquote 2: any excess periodate was quenched by addition of 20 mL glycerine (added under stirring and stirred for 3 h at rt). After the excess periodate was decomposed the product was washed by filtration. For aliquot 3: the product was washed by centrifugation using H2O x 6. For aliquot 4: the product was washed by filtration using H ₂ 0. All four aliquots produced bead-shaped DAC.

Example 5 - Preparation of DAC beads under unbuffered conditions

Cladophora cellulose, 3 g in 300 mL of deionized water was mixed with 20 g sodium metaperiodate (about 5 mol per mol of anhydroglucose units) dissolved in 300 mL deionized water. The periodate-containing reaction mixture was carefully wrapped in aluminum foil to avoid light. The reaction mixture was shaken by an orbital shaker at room temperature in the dark for 10 days. The reaction mixture was quenched via the addition of ethylene glycol and washed repeatedly with water to provide pure bead- shaped DAC material.

Example 6 - Preparation of DAC beads under unbuffered conditions without addition of 1-propanol

Cladophora cellulose, 2 g in 100 mL of deionized water was mixed with 13.2 g sodium metaperiodate (about 5 mol per mol of anhydroglucose units) dissolved in 100 mL deionized water. The periodate-containing reaction mixture was carefully wrapped in aluminum foil to avoid light. The reaction mixture was vigorously stirred at room temperature in the dark for 10 days. The product of the reaction yielded beads. Example 7 - Preparation of DAC beads using bacterial cellulose

Never-dried bacterial cellulose, corresponding to a dry weight of 2 g, was dissolved in 100 mL of water and mixed with 26.4 g sodium metaperiodate dissolved in 100 mL of water (about 10 mol per mol of glucopyranose). The periodate-containing reaction mixture was carefully wrapped in aluminum foil to avoid light and 20 mL of 1-propanol was added to the reaction mixture to serve as a radical scavenger. The reaction mixture was vigorously stirred at room temperature in the dark for 22 days. The reaction was quenched via the addition of ethylene glycol and washed by centrifugation using H ₂ 0 x 6 to provide pure bead-shaped product. Figure 1 B shows a SEM micrograph of the DAC beads formed after 22 days of periodate treatment.

Example 8 - Reductive amination of amines The DAC beads prepared in Example 1 were reacted with each of 1 ,2-diaminoethane, 1 ,3-diaminopropane, 1 ,4-diaminobutane, 1 ,5-diaminopentane, 1 ,6-diaminohexane, 1 ,7- diaminoheptane, bis-(4-diaminophenyl) ether aniline, o-phenylenediamine, m- phenylenediamine, p-phenylenediamine, 1 , 10-phenanthrolin-5-amine, 3-(amino methyl)pyridine, tryptophan, cysteine and hydroxylamine according to the following general procedure: To a stirred 100 mL RB-flask was added never dried DAC (corresponding to a dry weight of 100 mg), 40 mL buffer and amine. The reaction mixture was stirred at room temperature for 24 h. The product was washed by centrifugation using H ₂ 0 x 6. The resulting product was treated with sodium borohydride (1.2 equiv.) and reduction was performed during 2 h. The crude product was washed by centrifugation using H2O x 6. Figure 1 C exemplifies Schiff base coupled DAC beads, where Figure 1 C shows coupling with hydroxylamine.

Example 9 - Preparation of sulfonated DAC beads To a stirred 250 mL RB-flask was added never dried DAC from Example 1 (corresponding to a dry weight of 2 g), and 100 mL 0.5 M sodium bisulfite solution. The reaction mixture was stirred at room temperature for 24 h. The product was washed by centrifugation using EtOH x 6. Example 10 - Preparation of thioester DAC beads

To a stirred 25 mL RB-flask was added never dried DAC from Example 1 (corresponding to a dry weight of 405 mg), CuCI 2.475 mg, ferf-Butyl,hydroperoxide-solution (aq.) 256 μΙ_, ethyl-2-mercaptopropionate 134 mg and H2O 3 mL. The reaction mixture was refluxed for 1 h and washed with H2O x 6.

Example 1 1 - Preparation of porous DAC beads The DAC beads prepared in Example 1 were reacted with 1 ,7-diaminoheptane, according to the following procedure: To a stirred 100 mL RB-flask was added never dried DAC (corresponding to a dry weight of 100 mg), 40 mL buffer and 40 mg 1 ,7- diaminoheptane. The reaction mixture was stirred at room temperature for 24 h. The product was washed by centrifugation using H ₂ 0 x 6. The resulting imine product was treated with sodium borohydride (1.2 equiv.) and reduction was performed during 2 h. The crude product was washed by centrifugation using H ₂ 0 x 6 and EtOH x 2 and dried in air. Figure 1 D shows a SEM micrograph of the 1 ,7-diaminoheptane coupled DAC beads. The diamino coupled DAC beads were stable in alkaline solution (1 M NaOH). Example 12 - Preparation of DAC beads at pH 5.5 with regeneration of periodate

Cladophora cellulose, 20 g in 1000 mL of acetate buffer pH 5.5 was mixed with 132 g sodium metaperiodate (about 5 mol per mol of anhydroglucose units) dissolved in 1000 mL acetate buffer pH 5.5. The periodate-containing reaction mixture was carefully wrapped in aluminum foil to avoid light and 200 mL of 1-propanol was added to the reaction mixture to serve as a radical scavenger. The reaction mixture was vigorously stirred at room temperature in the dark for 10 days. The product was carefully washed with water and the liquid content was collected and reduced to a volume of 1 L via rotary evaporation. To the liquid mixture was added 195 g Oxone in the course of 3 h. The pH was controlled at 6-7 via the addition of sodium hydroxide solution. During the reaction a white precipitate was formed, which was filtered off and identified as periodate.

Example 13 - Preparation of DAC beads with 1 equiv. of sodium metaperiodate To a solution of sodium metaperiodate, 10.7 g in 100 mL H ₂ 0 was added Cladophora cellulose, 8.1 g (about 1 mol periodate per mol of anhydroglucose units). The periodate- containing reaction mixture was carefully wrapped in aluminum foil to avoid light. The reaction mixture was vigorously stirred at room temperature in the dark for 10 days. The reaction mixture was quenched via the addition of ethylene glycol and washed repeatedly with water to provide pure bead-shaped DAC material.

Example 14 - DAC beads functionalized with quaternary ammonium alkylamine

The DAC beads prepared in Example 1 were reacted with 1 ,7-diaminoheptane, according to the following procedure: To a stirred 100 mL RB-flask was added never dried DAC (corresponding to a dry weight of 100 mg), 40 mL buffer and 20 mg 1 ,7- diaminoheptane. The reaction mixture was stirred at room temperature for 24 h. The product was washed by centrifugation using H2O x 6. The resulting product was dispersed in 40 mL buffer solution and 100 mg (2-Aminoethyl)trimethylammonium chloride hydrochloride was added. The reaction mixture was stirred at room temperature for 24 h. The product was washed by centrifugation using H2O X 6. The resulting imine product was treated with sodium borohydride (1.2 equiv.) and reduction was performed during 2 h. The crude product was washed by centrifugation using H ₂ 0 x 6 and EtOH x 2 and dried in air. Example 15 - DAC beads functionalized with propanesulfonic acid

The DAC beads prepared in Example 1 were reacted with 1 ,7-diaminoheptane, according to the following procedure: To a stirred 100 mL RB-flask was added never dried DAC (corresponding to a dry weight of 100 mg), 40 mL buffer and 20 mg 1 ,7- diaminoheptane. The reaction mixture was stirred at room temperature for 24 h. The product was washed by centrifugation using H ₂ 0 x 6. The resulting product was dispersed in 40 mL buffer solution and 85 mg 3-Amino-1 -propanesulfonic acid was added. The reaction mixture was stirred at room temperature for 24 h. The product was washed by centrifugation using H ₂ 0 x 6. The resulting imine product was treated with sodium borohydride (1.2 equiv.) and reduction was performed during 2 h. The crude product was washed by centrifugation using H ₂ 0 x 6 and EtOH x 2 and dried in air.

Example 16 - Determination of aldehyde content The DAC samples were transformed to oximes via Schiff base reactions with hydroxylamine according to literature procedure (Kim et al., Biomacromolecules, Vol. 1 pp. 488-492 (2000)) and analyzed for elemental composition (C, H and N) according to the following procedure. To a stirred 100 mL RB-flask was added never dried DAC (corresponding to a dry weight of 100 mg), 40 mL acetate buffer (pH 4.5) and 1.65 mL hydroxylamine solution (ag. 50 wt%) was added. The reaction mixture was stirred at room temperature for 24 h. The product was thoroughly washed with water and dried under reduced pressure prior to elemental analysis. The term "degree of oxidation" (D.O.) represents the ratio of 2,3-alcohols in the anhydroglucose units that has been transformed into their corresponding aldehydes. The highest degree of oxidation, viz. 100%, corresponds to all anhydroglucose units being converted to the corresponding non-cyclic 2,3-dialdehyde structure, which would correspond to approximately 2.5 mmol of aldehyde groups per gram of cellulose.